Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro

Background—Hepatic stellate cellsplay a key role in the pathogenesis of hepatic fibrosis.
Aims—To examine the inhibitoryeffect of oestradiol on stellate cell activation.
Methods—In vivo, hepatic fibrosiswas induced in rats by dimethylnitrosamine or pig serum. In vitro, ratstellate cells were activated by contact with plastic dishes resultingin their transformation into myofibroblast-like cells.
Results—In the dimethylnitrosamineand pig serum models, treatment with oestradiol at gestation relateddoses resulted in a dose dependent suppression of hepatic fibrosis withrestored content of hepatic retinyl palmitate, reduced collagencontent, lower areas of stellate cells which express α smooth muscleactin (α-SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, α-SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation.
Conclusion—The antifibrogenic roleof oestradiol in the liver may contribute to the sex associateddifferences in the progression from hepatic fibrosis to cirrhosis.

Keywords:hepatic stellate cells; hepatic fibrosis; oestradiol; α smooth muscle actin; retinyl palmitate

     

姜黄素对肝星状细胞增殖及分泌细胞外基质的影响

观察姜黄素对大鼠肝星状细胞 (HSC)增殖及分泌细胞外基质的影响。用不同浓度的姜黄素处理大鼠肝星状细胞 ;以MTT比色法测定细胞的增殖 ;放射免疫法测定透明质酸和层粘连蛋白 ;酶联免疫吸附法 (ELISA)测定Ⅰ型胶原的水平。姜黄素可剂量依赖性地抑制HSC增殖 ,不同程度抑制Ⅰ型胶原、透明质酸和层粘连蛋白分泌。姜黄素可显著抑制HSC的增殖及分泌细胞外基质。     

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.     

Characteristics of the cell proliferation profile of activated rat hepatic stellate cells in vitro in contrast to their fibrogenesis activity

Purpose. Alterations in the kinetics of hepatic stellate cells (HSCs) after the cells are activated once have not been well documented. We investigated the characteristic profiles of cell proliferation of once-activated HSCs in contrast to the in fibrogenesis activity. Methods. HSCs from male Wistar rats were submitted to primary culture for 14 days and to secondary culture for 7 days. The potential for cell proliferation was evaluated by the number of the cells in G2/M phase, based on flow cytometric analysis of the cell cycle. The fibrogenesis activity was assessed by Northern blot analysis of the expression of type I and type III procollagen mRNA. Results. The number of HSCs in G2/M phase was maintained at a low level in primary culture after 6 days, while a significantly (P < 0.05) elevated number of HSCs in G2/M phase was observed on days 3 to 4. In secondary culture, the number of HSCs in G2/M phase was also consecutively maintained at a decreased level. By contrast, HSCs showed progressively increased type I and type III procollagen mRNA expression during the experimental periods of primary culture. Conclusions. These results clearly demonstrated consecutively decreased proliferative activity, evaluated by the potential for cell mitogenesis, in once-activated HSCs, in contrast to their progressively increased fibrogenesis activity. Received: April 12, 2000 / Accepted: December 22, 2000     

β-榄香烯对肝星状细胞凋亡相关蛋白Bcl-2家族的影响

[目的]研究β-榄香烯对肝星状细胞(HSC)凋亡及凋亡相关蛋白的影响。[方法]传代培养的大鼠HSC系HSC-T6与不同剂量β-榄香烯(终浓度为10、5、2.5 mg/L)共同培养48 h后,应用Annexin-V-碘化丙锭染色检测β-榄香烯对HSC凋亡的影响,免疫组化检测凋亡相关蛋白BcL-2/Bax。[结果]β-榄香烯可使HSC凋亡率明显增多,同时可使凋亡抑制分子Bcl-2表达下调,促凋亡分子Bax表达增强(P<0.01)。[结论]β-榄香烯可通过下调Bcl-2/Bax比率,促进HSC凋亡。     

龙血素B对肝星状细胞增殖及细胞外基质分泌的影响

目的观察龙血素B对大鼠肝星状细胞(HSC)增殖及细胞外基质分泌的影响。方法用不同浓度的龙血素B处理大鼠HSC;MTT法计算药物的抑制率;放射免疫法测定细胞上清中透明质酸(HA)、层粘连蛋白(LN)和Ⅳ型胶原(Ⅳ-C)。结果随药物浓度的升高,龙血素B对HSC-T6增殖的抑制作用逐渐增强,有明显的量效关系。细胞上清中HA、LN和Ⅳ-C的含量与对照组相比降低,其中龙血素B浓度为0.300μg/μL时降低最明显。结论龙血素B可抑制HSC-T6增殖,并不同程度抑制HA、LN和Ⅳ-C的分泌。     

Activated hepatic stellate cells participate in liver fibrosis in a patient with transfusional iron overload

We describe liver fibrosis caused by iron overload after a long history of blood transfusion in a patient with chronic renal failure. Pertinent laboratory data were: serum (s)-Fe 148 μg/dl; unsaturated iron binding capacity (UIBC) 14 μg/dl; s-ferritin 9350 ng/ml; human leukocyte antigen (HLA) A2, A24, B39, B55, Cw1, Cw7. Computed tomography revealed a high density in the liver, and laparoscopy revealed a brown liver. Liver histology showed bridging fibrosis from portal tracts. A heavy iron deposit was seen in Kupffer cells as well as in hepatocytes surrounded by fibrosis around the portal tracts. Immunocytochemistry revealed α-smooth muscle actin in many stellate cells distributed along the fibrotic area, and electron microscopy revealed infiltrating myofibroblastic stellate cells coexisting with collagen fibers around degenerated hepatocytes containing iron deposits. The findings are consistent with the notion that stellate cells play an important role in liver fibrogenesis in both genetic and transfusional iron overload hemochromatosis. Received Aug. 15, 1997; accepted Feb. 27, 1998     

Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies

Activation of hepatic stellate cells (HSCs) is the dominant event in liver fibrosis. The early events in the organization of HSC activation have been termed initiation. Initiation encompasses rapid changes in gene expression and phenotype that render the cells responsive to cytokines and other local stimuli. Cellular responses following initiation are termed perpetuation, which encompasses those cellular events that amplify the activated phenotype through enhanced growth factor expression and responsiveness. Multiple cells and cytokines play a part in the regulation of HSC activation. HSC activation consists of discrete phenotype responses, mainly proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis and retinoid loss. Currently, antifibrotic therapeutic strategies include inhibition of HSC proliferation or stimulation of HSC apoptosis, downregulation of collagen production or promotion of its degradation, administration of cytokines, and infusion of mesenchymal stem cells. In this review, we summarize the latest advances in our understanding of the mechanisms of HSC activation and possible antifibrotic therapeutic strategies.     

Somatostatin inhibits pro-inflammatory cytokine secretion from rat hepatic stellate cells.

BACKGROUND/AIMS: Activated hepatic stellate cells (HSCs) have been implicated in hepatic fibrosis. Somatostatin (SOM) has an immunomodulatory role. The aim of this study was to assess the secretion of pro-inflammatory cytokines by HSCs and to determine the effect of SOM on the secretion of these mediators. METHODS: Activated rat HSCs were evaluated for their secretion of IL-1beta, IL-8, and TNF-alpha using ELISA. RNA protection assay was used to determine cytokine mRNA levels. The expression of chemokine and cytokine mRNA and the secretion of these mediators were assessed following incubation with SOM or octreotide. RESULTS: HSCs spontaneously secreted IL-1beta, IL-8, and TNF-alpha. This secretion was augmented following stimulation by IL-1beta and TNF-alpha. SOM inhibited the spontaneous and TNF-alpha-induced secretion of IL-1beta, IL-8, and TNF-alpha and suppressed the expression of IL-1beta and TNF-alpha mRNA. Octreotide suppressed the secretion of IL-1beta and IL-8. CONCLUSIONS: These observations indicate that SOM exerts an inhibitory immunomodulatory effect on HSCs.     

抗纤软肝颗粒对大鼠肝星状细胞增殖的影响

目的:探讨抗纤软肝颗粒(KXRG)对大鼠肝星状细胞(HSC)增殖的影响。方法:传代培养的HSC-T6(肝星状细胞系)与KXRG(1.25mg/ml、2.5mg/ml、5mg/ml)及其药物血清(5%、10%、20%)共同培养72小时后,应用MTT法测定细胞增殖、流式细胞仪测定HSC细胞周期各时相的DNA含量。结果:KXRG及药物血清均能显著抑制HSC的增殖,使细胞周期阻滞于S期,且2.5mg/ml药物和10%药物血清作用最强(P0.01)。结论:KXRG通过抑制HSC的活化,阻滞其细胞周期的正常进行,进而抑制HSC增殖,从而起到抗肝纤维化的作用。     

去甲肾上腺素对大鼠肝星状细胞作用的实验研究

目的 探讨交感神经系统在肝纤维化发生和发展中的作用. 方法采用免疫荧光和RT-PCR检测体外培养的肝星状细胞(HSC)中α1、β2-肾上腺素能受体的表达;用四甲基偶氮唑盐法检测不同浓度的去甲肾上腺素(NE)对HSC增殖活性的影响.同时用RT-PCR检测受NE作用后HSC的活化指标胶原蛋白-1、转化生长因子β(TGF β)及α-平滑肌动蛋白(α-SMA)的表达.用高效液相色谱-电化学法测定活化的HSC中交感神经递质NE的水平. 结果α1和β2-肾上腺素能受体表达于HSC的胞膜和胞质内;NE可呈剂量依赖性地促进HSC增殖,在浓度为100μmol/L时达到最大效应,F=140.464,P<0.05,差异有统计学意义.以NE 100 μmol/L作用细胞24h后,可显著促进反应HSC活化的指标上升,胶原蛋白-1表达为0.3022±0.0610,TGF β表达为2.2080±0.2151,α-SMA mRNA表达为0.5469±0.0108,与对照组胶原蛋白-1(0.1040±0.0556)、TGF β(1.1190±0.0070)、α-SMA mRNA表达(0.0759±0.0449)比较,t值分别为-4.160、-8.763和-17.651,P值均<0.05,差异均有统计学意义.HSC可以合成并释放NE,且受血小板衍生生长因子(10ng/ml)刺激后HSC中NE含量为(14.24±0.21)ng/ml,对照组为(11.34±0.15)ng/ml,两组比较,t=-32.907,P<0.05,差异有统计意义.结论 抑制交感神经系统使HSC活性降低对临床上治疗肝纤维化有一定的指导意义.     

Arg-gly-asp-mannose-6-phosphate inhibits activation and proliferation of hepatic stellate cells in vitro

AIM: To investigate the effect of arg-gly-asp-mannose-6 phosphate (RGD-M6P) on the activation and proliferation of primary hepatic stellate cells in vitro. METHODS: Hepatic stellate cells (HSCs) were isolated from rats by in situ collagenase perfusion of liver and 18% Nycodenz gradient centrifugation and cultured on uncoated plastic plates for 24 h with DMEM containing 10% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, HSCs were cultured in 2% FBS/DMEM with transforming growth factor beta1, M6P, RGD, or RGD-M6P, respectively. Cell morphology was observed under inverted microscope, smooth muscle alpha-actin (alpha-SMA) was detected by immunocytochemistry, type III procollagen (PC III) in supernatant was determined by radioimmunoassay, and the proliferation rate of HSCs was assessed by flow cytometry. RESULTS: RGD-M6P significantly inhibited the morphological transformation and the alpha-SMA and PC III expressions of HSCs in vitro and also dramatically prevented the proliferation of HSCs in vitro. Such effects were remarkably different from those of RGD or M6P. CONCLUSION: The new compound, RGD-M6P, which has a dramatic effect on primary cultured HSCs in vitro, can inhibit the transformation of HSCs in culture caused by TGFbeta1, suppresses the expression of PC III and decreases proliferation rate of HSC. RGD-M6P can be applied as a selective drug carrier targeting at HSCs, which may be a new approach to the prevention and treatment of liver fibrosis.     

肝星状细胞在逆转肝纤维化中的作用

肝纤维化是一种能够导致门静脉高压、肝硬化、肝衰竭的严重疾病。已经发现肝星状细胞的活化是引起肝纤维化的中心环节,因此抑制肝星状细胞的活化、加速肝星状细胞的清除有望逆转肝纤维化。本文将对活化的肝星状细胞的凋亡、衰老以及清除的研究进展作综述,阐明肝星状细胞在肝纤维化过程中所起的作用及相关机制。     

Platelet-derived adenosine 5'-triphosphate suppresses activation of human hepatic stellate cell: In vitro study

Aim: Activated hepatic stellate cells (HSC) play a critical role in liver fibrosis. Suppressing abnormal function of HSC or reversion from activated to quiescent form is a hopeful treatment for liver cirrhosis. The interaction between platelets and HSC remains unknown although platelets go through hepatic sinusoids surrounded by HSC. This study aimed at clarifying the hypothesis that platelets control activation of HSC. Methods: We used human platelets, platelet extracts, and primary or immortalized human HSC. We examined the effect of platelets on the activation, DNA synthesis, type I collagen production, and fibrosis-relating gene expressions of HSC. We investigated what suppressed activation of HSC within platelets and examined the mechanism of controlling activation in vitro. Results: Platelets and platelet extracts suppressed activation of HSC. Platelets decreased type I collagen production without affecting DNA synthesis. Platelets increased the expression of matrix metallopeptidase 1. As platelet extracts co-cultured with an enzyme of degrading adenosine 5'-triphosphate (ATP) suppressed activation, we detected adenine nucleotides within platelets or on their surfaces and confirmed the degradation of adenine nucleotides by HSC and the production of adenosine. Adenosine and platelets increased the intracellular cyclic adenosine 5'-monophosphate (cAMP), which is important in quiescent HSC. A great amount of adenosine and ATP also suppressed activation of HSC. Conclusion: Activation of human HSC is suppressed by human platelets or platelet-derived ATP via adenosine-cAMP signaling pathway in vitro. Therefore, platelets have the possibility to be used in the treatment of liver cirrhosis.     

肝星状细胞激活与信号转导

肝纤维化是多种慢性肝病向肝硬化发展的必经阶段,是所有慢性肝病的共同病理基础.目前认为肝星状细胞的激活是肝纤维化形成的关键,各种致病因素作用下引起肝损伤,释放多种细胞因子,如转化生长因子β、血管紧张素、瘦素,通过各种信号转导使肝星状细胞激活.本文就肝星状细胞激活过程中一些重要的膜受体、核受体信号转导途径及其研究进展作一综述.